The present invention related to a CDNA clone, designated to PepCYP (pepper cytochrome P450 gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof
Colletotrichum gloeosporioides (Penz.) is the casual agent of anthracnose diseases on fruit crops (Daykin 1984; Dodd et al. 1991; Prusky et al. 1991) such as pepper (Capsicum annuum L.) (Kim et al. 1986; Manandhar et al. 1995). The infection of C. gloeosporioides is achieved through conidium germination and formation of appressorium and infection hyphae which are necessary for subsequent cuticular penetration (Bailey et al. 1992). In the avocado-C. gloeospoioides interaction, conidium germination and appressorium formation were similar on both unripe-resistant and ripe-susceptible fruits (Prusky and Saka 1989; Prusky et al. 1991). In the pepper and C. capsici pathosystem, germination and appressorium formation were higher on the unripe-resistant pepper fruit than on the ripe-susceptible fruit (Adikaram et al. 1983). The germination of C. musae was similar on both ripe-susceptible and unripe-resistant banana fruits, but the appressorium formation was stimulated on the unripe fruit (Swinburne 1976). Taken together, in these pathosystems, although conidium germination and appressorium formation are a prerequisite to infect the host plant, they may not be important factors to determine resistant or susceptible interactions between the host fruit of pre- or post-ripening stages and the Colletotrichum fungus. In contrast, plant responses to fungal morphogenesis during fruit ripening may be more important to determine resistant or susceptible interactions.
In Colletotrichum fungi, the topography of the plant surface was thought to be a physical signal for inducing appressorium formation (Staples and Macko 1980). The surface wax of avocado fruit was reported to act as a chemical signal for appressorium formation in C. gloeosporioides (Podila et al. 1993). In addition, the genes expressed during the appressorium formation of C. gloeosporioides induced by the host surface wax were cloned (Hwang and Kollatukudy 1995). However, plant responses to the conidium germination and appressorium formation of C. gloeosporioides have not been studied.
In a previous study, we found that an isolate of C. gloeosporioides showed an incompatible interaction with the ripe-red pepper fruit and a compatible interaction with the unripe-mature-green fruit (Oh et al. 1998), even though ripe fruits are generally susceptible to pathogen infection (Prusky et al. 1991; Swinburn 1983). In this pathosystem, higher levels of appressorium and infection hypha formation, and longer infection hypha were observed on the unripe fruit than on the ripe fruit in the early fungal infection step (Kim et al. 1999). A typical sunken necrosis was detected only in the unripe fruit, but not in the ripe fruit at 5 days after inoculation. Based on these data of the fungal morphogenesis and symptom development, we studied the gene expression in the pepper-C. gloeosporioides interaction.
By using a mRNA differential display method, we isolated several cDNAs that are differentially induced in the ripe fruit, but not in the unripe fruit after fungal infection. In this study, we report the characterization of one of these cDNAs encoding cytochrome P450 protein (PepCYP). To our knowledge, PepCYP is the first cytochrome P450 gene that accumulates to high levels in an incompatible plant-fungus interaction. The expression of PepCYP gene was examined based on fungal behaviors in the initial infection process and on symptom development during the differential interactions. We found that the PepCYP gene is induced by wounding or exogenous jasmonic acid during ripening. A possible role of the PepCYP in pepper fruits against pathogen infection during ripening is proposed.
The present invention relates to a cDNA clone, designated to a pepper cytochrome P450 gene, PepCYP, the sequence of which is depicted in SEQ ID No. 1. The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. By using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein SEQ ID NO:2 homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in compatible interaction, and then remains elevated in the incompatible interaction. However, in the compatible interaction the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 h and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process. The PepCYP gene can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with these genes. In addition, the PepCYP gene of this invention can be also used to produce transgenic plants that exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematode, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.